Nocistatin was recently isolated from bovine brain and shown to block hyperalgesia and allodynia induced by nociceptin and prostaglandin (PG) E2. The counterparts of human, rat and mouse are deduced from their precursor prepronociceptin to be 30, 35, and 41 residue peptide respectively. To identify these mature forms of nocistatin, three peptides were synthesized and a detection program for nocistatin was developed, using high pressure liquid chromatography (HPLC) along with specific radioimmunoassay (RIA). Nocistatin extracted from human, rat and mouse brain were subjected to HPLC and nocistatin-like immunoreactivity (NST-IR) was determined. All three species showed two NST-IR peaks, one of which coincided with that of the corresponding putative nocistatin. The same NST-IR was also detected in human cerebrospinal fluid (CSF).